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Hydroxylated isoprenoid GDGTs (OH‐GDGTs) have emerged as a novel tool for reconstructing sea surface temperatures. However, when using marine OH‐GDGT calibration in lacustrine settings, it leads to a significant overestimation of temperatures, emphasizing the necessity for a thorough examination of OH‐GDGTs in lakes. Here, we investigated OH‐GDGT distributions in surface sediments from 65 lakes in West China and compiled published Asian lake and global marine OH‐GDGT data sets. Among all GDGT‐based indices, RI‐OH showed the strongest correlation with temperature across Asian lakes. The RI‐OH value was higher in lakes than in marine sediments, likely due to differences in the composition of Group 1.1a thaumarchaeotal species between the two settings. The first RI‐OH temperature calibration for lakes was developed and it addressed the issue of temperature overestimation when applied to both water column and sediment core, highlighting the potential of OH‐GDGTs as a new terrestrial paleothermometer. Plain Language Summary: Reconstruction of past terrestrial temperatures plays a crucial role in understanding modern global warming and projecting future climate patterns, yet reliable terrestrial temperature proxies remain limited. In this study, we present compelling evidence that the ring index of OH‐GDGTs, expressed as RI‐OH, is predominantly influenced by temperature in lacustrine settings, regardless of the wide range of physical and chemical characteristics of the lakes. We developed the first lacustrine RI‐OH‐temperature calibration and successfully applied it to a water column and a sediment core. These findings suggest that RI‐OH can be used as a novel paleothermometer in lacustrine settings. Key Points: OH‐GDGTs were first investigated in lacustrine settings along a wide temperature gradientTemperature was the most important factor affecting OH‐GDGT distributions in lacustrine settingsThe first calibration in lacustrine settings was established and successfully applied to a water column and a sediment core [ABSTRACT FROM AUTHOR]